The effects of dietary supplements for modifying cattle digestion and metabolism are typically measured after one or more weeks of adaptation. Consequently, how quickly the effects occur remains unknown. The long-term efficacy of Acacia mearnssii bark tannins (Acacia) on methane mitigation has been previously demonstrated. The present study, therefore, investigated the time it took for the extract to affect methane emissions and milk fatty acid profiles. Twenty lactating Brown Swiss dairy cows, categorized as 10 low- and 10 high methane emitters (average difference in methane yield: 0.10 of total), were housed in respiration chambers for 4 days. A control diet consisting of a mixed ration supplemented with grass pellets and concentrate pellets was fed initially (Day 0). The original pellets were then replaced with pellets containing 141 g Acacia/kg, providing 30 g Acacia/kg of dietary dry matter (DM) (Days 1–3). Methane emissions were measured every 10 min and gas chromatography was used to analyze individual fatty acids from daily milk samples. A significant decline in methane production was detected 20 min after starting supplementation, with methane production (g/day) and methane yield (g/kg DM intake) decreasing in a linear fashion from Day 0 to Day 3 by up to proportionately 0.18 and 0.16, respectively. Additionally, changes in proportions of various milk fatty acids occurred within 3 days of Acacia feeding. Using stepwise multiple regression analysis, several milk fatty acids were identified as being related to methane emissions. Applicable equations from the literature also showed relationships to methane emissions of high and low emitters as well as to the Acacia diet effect on methane emissions. The equations with close relationships all included minor and nondietary milk fatty acids like odd-chain fatty acids. These specific fatty acids originate from rumen microbial activity. In conclusion, A. mearnsii extract is an immediately acting methane mitigating supplement. Certain milk fatty acids are related to methane emission in dairy cows and may assist in determining whether and when tanniferous supplements will act against enteric methane formation.